Lambda phosphatase

Approximately 0.8 mg of lyophilized, ¹⁵N‐labeled pS16M179 was resuspended in 250 μl of water and allowed to sit at room temperature for 24 h. This solution was added to a 250 μl solution containing 50 mM TrisHCl, pH 8.5, while gently vortexing. Fifty μl of 10× Reaction Buffer (500 mM HEPES, 1 mM EGTA, 50 mM dithiothreiotol, 0.1% BRIJ 35), 50 μl of 10× MgCl₂ (20 mM), and 5 μl of lambda phosphatase (400 units/uL, Santa Cruz Biotechnology, Dallas, TX) was added to this solution that had a final pH of 7.8. The reaction solution was incubated overnight at 303 K. Prior to purification by reverse phase chromatography as described above, an equal volume of reverse phase Buffer A was added to the solution and the pH adjusted to ~3 with 1 μl additions of TFA. A similar experiment was performed on a 0.5 mM sample of unlabeled pS16M179 in a Shigemi NMR tube (260 μl) under the same buffer conditions except the pH was adjusted to 7.1 prior to the addition of 5 μl of lambda phosphatase.

HEK293 cells latently infected with BRRF2-knockout EBV were transfected with pcDNABZLF1 together with expression vector for wild-type or mutant FLAG-tagged BRRF2. After one day, cells were lysed in phosphatase buffer (Santa Cruz). Lysed samples were reacted with or without Lambda phosphatase (Santa Cruz). Protein samples were separated on a 7.5% Phos-tag gel (Wako chemicals). After electrophoresis, the Phos-tag gel was washed with transfer buffer containing EDTA to remove zinc ions from the gel, according to the manufacturer’s instructions, followed by immunoblotting as specified above.